Synthesis of Oligomers in Arrays

ABSTRACT

Systems, including apparatus and methods, for synthesis of oligomers in arrays.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/172,753 filed Jun. 30, 2005, which claims the benefit under 35 USC§119(e) of U.S. Provisional Application No. 60/584,524 filed Jun. 30,2004, both of which are incorporated herein by reference in theirentirety.

INTRODUCTION

Oligomers are chemical compounds, such as oligonucleotides or peptides,that include a covalently linked chain of individual subunits. Theidentity of each individual subunit and the sequence of the individualsubunits within the chain generally define the chemical and biologicalproperties of each oligomer. In particular, a small change in thechemical structure of an oligomer, such as a single nucleotide change inan oligonucleotide, can impart quite distinct biological properties tothe oligomer. Accordingly, large sets of oligomers can be synthesizedfor use in various clinical and research applications.

SUMMARY

The present teachings provide systems, including apparatus and methods,for synthesis of oligomers in arrays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary system for solid-phasesynthesis of oligomers in an array using a synthesis support devicehaving a porous member that defines an array of addressable sites, inaccordance with aspects of the present teachings.

FIG. 2 is a schematic view of the synthesis support device of FIG. 1, inaccordance with aspects of the present teachings.

FIG. 3 is a view of another exemplary system for solid-phase synthesisof oligomers in an array using a synthesis support device having aporous member that defines an array of addressable sites, in accordancewith aspects of the present teachings.

FIG. 4 is a sectional view of the synthesis support device of FIG. 3,taken generally along line 4-4 of FIG. 3, in accordance with aspects ofthe present teachings.

FIG. 5 is fragmentary sectional view of an exemplary addressable site(and reaction compartment) from the synthesis device of FIG. 4, takengenerally from the region indicated at “5” in FIG. 4.

FIG. 6 is a series of fragmentary sectional views of configurations ofthe addressable site of FIG. 5 as the site is being addressed with afirst reagent in fluid isolation from other addressable sites of thesynthesis support device, and then with a second reagent in fluidcommunication with the other addressable sites, in accordance withaspects of the present teachings.

FIG. 7 is a series of fragmentary sectional views of configurations ofthe addressable site of FIG. 5 during post-synthesis processing ofoligomer populations synthesized on solid support surfaces of the site,in accordance with aspects of the present teachings.

FIG. 8 is a series of views of a porous member being processed so thatregions of the porous member are addressable selectively, in accordancewith aspects of the present teachings.

FIG. 9 is a series of view of structures produced during fabrication ofa channel (well) array for assembly with the selectively addressableporous member produced in FIG. 8 to form an array-defining portion of asynthesis support device, in accordance with aspects of the presentteachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

The present teachings provide systems, including apparatus and methods,for synthesis of oligomers in arrays. The apparatus can include asynthesis support device that defines an array of addressable sites (andreaction compartments). This array of sites can be defined by a porousmember having (1) a plurality of porous islands, and (2) a spacer ofdifferent surface energy (e.g., wettability) than the porous islandsthat separates the porous islands. For example, the islands can be morehydrophilic (or less hydrophobic) than the spacer. Accordingly, theislands can be addressed in isolation with smaller volumes of fluid,such as with selected reagents for oligomer synthesis, because thedifferent surface energy of the spacer can restrict lateral movement ofthe fluid away from the islands. In addition, the islands can beaddressed in fluid communication with one or more common reagents usinglarger volumes of fluid that can flood the islands and thereby overcomethe surface energy barrier created by the spacer. Methods ofsynthesizing oligomers using porous members also are disclosed.

Oligomers can be synthesized on reaction surfaces that overlap and/orare spaced from the porous islands. For example, the reaction surfacescan be provided by reaction compartments defined by the porous islandsand/or in reaction compartments disposed adjacent the porous islands,such as on adjoining channel walls and/or particles, among others.Accordingly, each oligomer can be synthesized on two or more distinctreaction surfaces that can provide separate populations of the oligomerfor the same or different purposes, such as structural analysis (e.g.,sequence verification and/or quality control) and/orexperimental/diagnostic use, among others. Therefore, the systems of thepresent teachings can offer (1) greater flexibility of solid-phaseoligomer synthesis and/or (2) synthesis of a larger amount of eacholigomer in a smaller area and/or with a smaller amount of reagents thansystems that synthesize oligomers on a nonporous planar surface, inmicroplate wells, and/or in separate columns.

FIG. 1 shows an exemplary system 20 for synthesizing oligomers in anarray. System 20 can include a synthesis support device or platform 22that defines an array of addressable sites and reaction compartments forsolid-phase oligomer synthesis. System 20 also can include a reagentdispenser 24, a flow controller 26, and/or a processor 28, among others.

Reagent dispenser 24 can be configured to dispense reagents selectivelyto individual sites of device 22 and/or to dispense one or more commonreagents nonselectively to many or all of the addressable sites of thesupport device.

Flow controller 26 can exert a pressure periodically or continuously onthe synthesis support device, to move reagents into and/or throughreaction compartments. Accordingly, the flow controller can include apump, a centrifuge, and/or pressurized gas, among others.

Processor 28 can be a data processor or controller configured to controland coordinate any suitable aspects of system 20. For example, processor28 can control positioning of the reagent dispenser, can select reagentsand volumes thereof to be dispensed, and/or can operate valves and/orpumps that effect dispensing of the selected reagents and volumes.Alternatively, or in addition, processor 28 can control operation offlow controller 26, such as selecting the times at which pressure isexerted on the synthesis support device, thereby controlling reaction(or reagent contact) times during oligomer synthesis.

FIG. 2 shows a schematic representation of synthesis support device 22.Support device 22 can include a porous member 42, reaction compartments44, and/or an adjoining chamber(s) or receiver compartment(s) 46, amongothers. Porous member 42 can define an array of addressable regions orsites, generally as an array of porous islands 48 separated by a spacerof different surface energy than the islands. Reagents can be receivedin each of the porous islands for placement in reaction compartments 44having reaction surfaces 50 for supporting solid-phase oligomersynthesis. Alternatively, or in addition, the reaction compartments canbe used to conduct liquid-phase oligomer synthesis, during whicholigomer intermediates are not coupled to a solid support surface.

Reaction compartments 44 and/or reaction surfaces 50 can be disposed inone-to-one correspondence with the porous islands. Accordingly, thereaction compartments and reaction surfaces can define arrays alignedwith, adjacent, and/or overlapping the array of porous islands 48included in the porous member. For example, reaction surfaces 50 can bein reaction compartments included in the porous islands and/or disposedadjacent the porous islands. In some examples, at least a portion ofeach reaction surface can be included in a reaction compartmentconfigured as a channel (a permeable well) adjoining each porous island.The channel can have a reaction surface defined by a wall of the channeland/or by a support matrix, such as particles, disposed in the channel,among others. The channel can be configured to receive a reagent from acorresponding porous island, to temporarily retain the reagent, and thento permit the reagent to be removed from the channel. The reagent can beremoved by fluid flow out of the channel into adjoining chamber 46,which can serve as a waste reservoir. Fluid flow can be created by apressure created by a flow controller in fluid communication with theadjoining chamber.

Further aspects of the present teachings are described in the followingsections, including (I) synthesis support devices, (II) reagentdispensers and reagents, (III) reagent removal mechanisms, and (IV)examples.

I. Synthesis Support Devices

The synthesis systems of the present teachings can include one or moresynthesis support devices. A synthesis support device generally includesany device having an array of reaction surfaces that are individuallyaddressable with selected reagents and capable of supporting oligomersduring their synthesis. Support devices can include an array portionwith a porous member and reaction compartments. The support device alsocan include a body portion or frame holding the array portion and atleast partially defining an adjoining chamber(s). Further aspects ofsupport devices are included in the following subsections, including (A)porous members, (B) reaction compartments, and (C) adjoining chambers.

A. Porous Members

A porous member, also termed a filtration device, generally includes anystructure having a plurality of pores permitting passage of fluidbetween opposing surfaces of the structure.

The porous member can have any suitable configuration and can be formedof any suitable material(s). The porous member can be one-piece(unitary), or it can be formed of or two or more distinct pieces. Insome examples, the porous member can be generally planar, with itsthickness substantially less than its length and width. The thickness ofthe porous member can be selected, for example, according to a desiredmechanical strength, ease of fabrication, a desired fluid capacity perunit area (according to pore size/density), and/or the like. In someexamples, the porous member can be nonplanar. The porous member can haveany suitable size and can be large enough to define an array of anysuitable size. The porous member can be formed of a material that is aconductor (including a metal or a semi-conductor, among others) or aninsulator. Exemplary materials that can substantially form the porousmember can include silicon, gallium, a metal(s), a metal alloy(s),plastic, ceramic, glass, and/or any combination thereof, among others.

The porous member can include a plurality of porous islands or porousregions separated by a spacer. Each porous island or region can beconfigured to permit fluid communication between opposing surfaces ofthe island. Accordingly, the porous island includes at least one pore(or opening), and generally a plurality of pores (or openings),configured to permit such fluid communication. The porous island canhave any suitable shape, including rectangular, circular, ovalloid,elliptical, etc.

Porous islands can be present in any suitable number and in any suitablearrangement. The porous islands can have a regular or irregular spacing.In exemplary embodiments, the porous islands are disposed in arectilinear arrangement of two or more rows and two or more columns.However, other suitable arrangements can include one row or one columnand can include a radial array, a staggered (e.g., hexagonal) array, anirregular array, and/or the like. In exemplary embodiments, the porousislands (and associated reaction compartments) can define an array ofabout 100 to 5,000 islands. In some examples, the array can correspondin spacing and arrangement to a microplate array, for example, amicroplate with 96, 384, or 1536 wells, among others. In such examples,the center-to-center spacing between islands can be about 9 mm, 4.5 mm,or 2.25 mm, among others, and the arrangement of islands can be an 8×12,16×24, or 32×48 rectangular array, among others.

The spacer can be any intervening material that at least substantiallyor completely separates the porous islands. The spacer can be joined tothe porous islands, for example, formed with the porous islands from asingle piece of material. Accordingly, the spacer can be porous and canhave pores that are similar (or different) in size and/or shape to thoseof the porous islands.

The porous islands and the spacer can have different surface energies toprovide differential wettabilities or surface tensions. Thesedifferences can be local and/or average differences, among others.Different surface energies, as used herein, can be a differentialaffinity for fluid sufficient or effective to restrict lateral movementor spreading of a liquid from an island to the spacer (and thus from anisland to an adjacent island). For example, the porous islands can havea higher surface energy than the spacer, so that the porous islands arerelatively hydrophilic and the spacer is relatively hydrophobic. A polarliquid (such as water or an aqueous solution) thus can be selectivelyaddressed to one (or more) of a plurality of hydrophilic islandsseparated by a hydrophobic spacer. This setup is particularly suitablefor synthesis of water-soluble oligomers or polymers, such as nucleicacids and proteins. Conversely, the porous islands can have a lowersurface energy than the spacer, so that the porous islands arerelatively hydrophobic and the spacer is relatively hydrophilic. Anonpolar liquid (such as an organic solvent) thus can be selectivelyaddressed to one (or more) of a plurality of hydrophobic islandsseparated by a hydrophilic spacer. This setup is particularly suitablefor synthesis of water-insoluble oligomers or polymers. Generally,relatively higher wettabilities imply a greater tendency for a fluid tospread on a solid surface and be imbibed by a porous surface, andrelatively lower wettabilities imply a lesser tendency for a fluid tospread on a solid surface and be imbibed by a porous surface. Thesurface energy can be a surface energy of an exterior surface of regionsof the porous member and/or of an interior surface defined by pores.Differences in surface energy between the islands and the spacer can becreated by differential surface modification/treatment of the islands orthe spacer and/or by forming the islands and spacer out of differentmaterials having different surface energies, among others. For example,one of the islands and spacer can be treated with a wetting agent,and/or the other of the islands and spacer can be treated with anonwetting or waterproofing agent, among others.

The relative affinity between a liquid and a solid surface can becharacterized by the contact angle between the liquid in contact withthe solid surface. This angle is determined by competition betweenliquid-liquid molecular forces and liquid-solid molecular forces, and sodepends in part on the particular solid and liquid involved, as well asthe smoothness and cleanliness of the surface. Generally, the smallerthe contact angle, the greater the affinity between the liquid and thesurface, and the more easily the liquid will penetrate pores formed bythe surface. In particular, in pores penetrated by capillary action, thefluid will rise (or extend) nearer the walls of the pore for contactangles less than 90 degrees (with 0 degrees being totally flat orspread), and the fluid will fall (or recede) nearer the walls for anglesgreater than 90 degrees (with 180 degrees between totally rounded up orspherical). However, the total penetration of liquid into the pore willbe determined by an interplay between contact angle, surface tension,and fluid density, among others. Thus, for a given liquid, the islandsand the spacer can be distinguished by different contact angles,typically less than 90 degrees for one, and greater than 90 degrees forthe other.

Pores, as used herein, are openings of any suitable diameter and shape.The pores can be macropores or nanopores. Macropores, as used herein,have an average diameter of equal to or greater than about onemicrometer, and nanopores have an average diameter of less than aboutone micrometer. Generally, capillary action will draw fluid more easilyinto small pores, and less easily into large pores, all other thingsbeing equal. The pores can be an interconnected set or network of poresor can follow separate paths between opposing surfaces of the porousmember. The pores can be present at any suitable density to achieve anysuitable permeability and fluid capacity of a porous member.

Pores can be created by any suitable process. The pores can be createdmechanically (e.g., using a drill), optically (e.g., using a laser),chemically (e.g., by wet-etching), electrically (e.g., by using anonporous member as an electrode), and/or as voids within an assembly offibers (such as a fiber filter), among others. In exemplary embodiments,the pores are formed by wet-etching a silicon wafer.

B. Reaction Compartments

The synthesis support device can include a plurality of reactioncompartments in fluid communication with, overlapping with, and/or atleast substantially coextensive with the porous islands. A reactioncompartment can include any space for receiving reagents and having areaction surface(s) to support synthesis of an oligomer(s) using thereagents.

The reaction compartment can be configured to hold fluid transiently andto permit removal of the fluid. Accordingly, the reaction compartmentcan be defined by and/or disposed adjacent a porous or permeablestructure. For example, the reaction compartment can be defined by aporous island of a porous member, with the walls of the pores beingreaction surfaces of the compartment. Alternatively, or in addition, thereaction compartment can be or include a space disposed adjacent theporous island.

The space adjacent the porous island can be a channel that permits fluidflowthrough. The channel can be configured to receive reagents at afirst end of the channel and to release at least a portion of thesereagents for removal at a second end of the channel. The first andsecond ends can be generally opposing. Accordingly, the first and secondends of the channel can be permeable, provided, for example, by (1) aporous member (such as a porous island thereof) and (2) a permeablelayer flanking the channel. Although the permeable layer can permitfluid flow, the permeable layer can be configured to reduce fluid flowso that reagents are retained at least transiently in the channel topermit chemical reactions to occur.

The channel can include or contain any suitable reaction surfaces tosupport oligomer synthesis. For example, the channel can have a walldefining a reaction surface. Alternatively, or in addition, the channelcan hold a matrix or discrete particles (such as beads) having reactionsurfaces. The particles can have any suitable size or shape and can beformed of any suitable material, including plastic (such as polystyrene,among others), glass (such as controlled-pore glass (CPG)), metal, etc.

The space adjacent the porous island, in some embodiments, can becreated by a well having a nonpermeable end/bottom wall. In theseembodiments, the reagents can be received and removed from the sameregion of the well.

The reaction surface can be any solid and/or persistent surface(including a gel) to which oligomer intermediates are connected duringoligomer synthesis. The reaction surface can provide a covalent linkageto oligomer intermediates (and oligomers). Accordingly, the reactionsurface can include a first reactive moiety configured to react to forma covalent bond with a second reactive moiety of an oligomer subunit orintermediate (or a precursor thereof). Exemplary pairs of first andsecond (or second and first) reactive moieties can be classified aselectrophilic and nucleophilic moieties, as presented in Table 1. Here,persistent means that the surface remains at least substantially intactor functional during the course of a surface-associated reaction.

TABLE 1 Chemically Reactive Moieties Electrophilic Moiety NucleophilicMoiety Resultant Covalent Linkage activated esters amines/anilinescarboxamides acyl azides amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazides hydrazones aldehydes or ketones hydroxylamines oximesaldehydes or ketones thiosemicarbazides thiosemicarbazones alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolthiophenols aryl halides amines aryl amines azindines thiols thioethersboronates glycols boronate esters carboxylic acids amines/anilinescarboxamides carboxylic acids alcohols esters carboxylic acidshydrazines hydrazides carbodiimides carboxylic acids N-acylureas oranhydrides diazoalkanes carboxylic acids esters epoxides thiolsthioethers haloacetamides thiols thioethers halotriazinesamines/anilines ammotriazines halotriazines alcohols/phenols triazinylethers imido esters amines/anilines amidines isocyanates amines/anilinesureas isocyanates alcohols/phenols urethanes isothiocyanatesamines/anilines thioureas maleimides thiols thioethers phosphoramiditesalcohols phosphite esters silyl halides alcohols silyl ethers sulfonateesters amines/anilines alkyl amines sulfonate esters thiols thioetherssulfonate esters carboxylic acids esters sulfonate esters alcoholsethers sulfonyl halides amines/anilines sulfonamides sulfonyl halidesphenols/alcohols sulfonate esters

Alternatively, or in addition, the reaction surface can provide anoncovalent association with oligomer intermediates (and completedoligomers), such as binding through a specific binding pair(antibody-antigen, receptor-ligand, enzyme-substrate, complementarynucleotide strands, etc.). The reaction surface for noncovalent orcovalent association can be any suitable external or internal surface(s)of the synthesis support device including the walls of a channel, thewalls of pores, the walls of a well, and/or the exterior/interiorsurface of particles, among others.

A reaction compartment can include two or more distinct reactionsurfaces permitting coupled oligomers to be separated during and/orafter their synthesis. The distinct reaction surfaces can be physicallyseparable, that is, disposed on separable structures. Alternatively, orin addition, the distinct reaction surfaces can be chemically distinctso that oligomers can be selectively removed from one or more of thereaction surfaces. Accordingly, distinct reaction surfaces of a reactioncompartment can provide oligomer coupling that is selectively sensitiveto any suitable uncoupling treatment, such as pH, heat, light, exposureto a particular chemical cleavage agent, etc.

C. Adjoining Chambers

A synthesis support device can have one or more chambers adjoining thearray portion of the support device. An adjoining chamber can besubstantially enclosed so that the chamber can hold a reduced (orincreased) pressure, to draw (push) fluid from the reaction compartmentsto (away from) the chamber. The adjoining chamber can be a singlechamber in fluid communication with an entire array of islands/reactioncompartments, for concurrent application of an increased or decreasedpressure to the islands/reaction compartments. Alternatively, theadjoining chamber can be a plurality of chambers, in fluid communicationwith individual islands/reaction compartments or subsets of two or moreislands/reaction compartments. Configuration of the adjoining chamber asa plurality of chambers can permit selective removal of fluid from asubset of the reaction compartments. In some examples, the synthesissupport device can include at least one adjoining chamber configured toreceive reagents from the reaction compartments, thereby serving as awaste reservoir.

The adjoining chamber can be created by a chamber structure adjoiningthe array portion (e.g., the porous member) of the support device. Thechamber structure can form a substantial seal against a surface of thearray portion, for example, against an upper surface, a lower surface,and/or a perimeter of the array portion. The chamber structure can bedisposed generally above and/or below the array portion of the synthesissupport device, for example, as a cover for the array portion and/or asa frame that supports the array portion.

II. Reagent Dispensers and Reagents

The synthesis systems of the present teachings can include one or morereagent dispensers configured to dispense reagents to a synthesissupport device. A reagent dispenser can include a dispense head, reagentreservoirs, conduits, valves, and/or pumps, among others. The reagentdispenser can dispense reagents using contact and/or noncontactmechanisms.

Each reagent dispenser can dispense reagents to addressable sites of thearray portion from one or more dispense heads, each having one or moredispense structures (such as dispense tips, among others). The dispensestructures of a dispense head can be fixed and/or movable in relation tothe addressable sites. The dispense structures can fixed or movablewithin a dispense head. In some embodiments, the systems describedherein can include two or more dispense heads that are movableindependently. Such dispense heads can be configured to dispense thesame reagents as each other (redundant dispense heads) or differentreagents. If the same or overlapping sets of reagents are dispensed bytwo or more dispense heads, corresponding dispense structures of thedispense heads can be connected to the same reagent reservoir ordifferent reservoirs. The use of two or more dispense heads (and/or theuse of two or more dispense tips per dispense head) can increasesynthesis throughput.

Reagent reservoirs disposed in fluid communication with the dispensestructures can store any suitable number of reagents. The dispensestructures can be connected in one-to one correspondence with a set ofreagent reservoirs. Alternatively, different reagent reservoirs can bein communication with the same dispense structure, to provide, forexample, mixed and/or alternate dispensing of reagents from thedifferent reagent reservoirs.

The reagent dispenser can include conduits, valves, and/or a pump topropel, guide, and/or restrict movement of reagents between the reagentreservoirs and the dispense structures. The conduits can define parallelpaths between the reagent reservoirs and the dispense structure.Alternatively, or in addition, the conduits can define a branchednetwork so that the same reagent reservoir can connect to a plurality ofdispense structures and/or so that a plurality of reagent reservoirs canconnect to the same dispense structure. The valves (or one valve) canopen and close the conduits and can be operable manually and/or througha controller. The open time for a valve can define the volume of reagentdispensed to a reaction vessel. The pump (or pumps) can be any mechanismthat propels reagents from the reagent reservoirs to the dispensestructures and/or that expels reagents from the dispense structures. Thepump can exert a pressure on reagents directly or on a compartment influid communication with the reagents. The pump can act to push and/orpull reagents during dispensing (e.g., by creating positive relativepressure within a dispense tip to push reagents out, or by creating anegative relative pressure outside the dispense tip to pull reagentsout, respectively, among others). Accordingly, the pump can be apositive-displacement pump (e.g., a syringe pump, a peristaltic pump, arotary pump, etc.), a vacuum pump, pressurized gas, a partial vacuum,and/or the like. In some embodiments, the gas (such as argon, amongothers) provided by the pump places reagents under a more inertenvironment, such as by reducing exposure to moisture, oxygen, etc. Insome embodiments, the dispenser can include nozzles configured todispense small volumes of some of or all of the reagents, for example,using inkjet technology, such as a piezoelectric dispense mechanismand/or a thermal dispense mechanism, among others.

The reagent dispenser can dispense any suitable reagents for synthesisof oligomers. Such reagents, generally termed oligomer reagents, caninclude oligomer components and ancillary reagents.

Oligomer components generally include any chemical compounds that arepartially or completely incorporated into oligomers during theirsynthesis, generally through covalent linkage. Oligomer components canbe configured so that reactive groups are protected, exposed, and/orcreated relative to parent compounds, as appropriate. An oligomercomponent can correspond to a portion or all of a subunit of anoligomer, a dimer of subunits, a trimer of subunits, etc. Exemplaryoligomer components include nucleic acid components, such asdeoxyribonucleotides, ribonucleotides, peptide nucleic acids, lockednucleic acids, or analogs, relatives, derivatives (e.g., phosphoramiditederivatives thereof, among others), or portions thereof. In someexamples, the oligomer components can include adenosine, cytidine,guanosine, and thymidine phosphoramidites held in individual reservoirs,to be addressed individually (or in combination) to reactioncompartments. Alternatively, or in addition, the oligomer components caninclude nucleotide derivatives with modified bases. Other exemplaryoligomer components include amino acids, or analogs, relatives,derivatives, or portions thereof, to form peptides or peptide analogs(peptidomimetics). Additional exemplary oligomer components can includecarbohydrates, lipids, metalorganic compounds, etc.

Ancillary reagents can include any other reagents that facilitateoligomer synthesis. Such ancillary reagents can include a solvent orfluid carrier, reagents for capping (protection of reactive groups),deprotection, oxidation, reduction, cyclization, washing, cleavage(uncoupling from a reaction surface), etc. In some embodiments, thefluid carrier can be or include acetonitrile. Alternatively, or inaddition, the fluid carrier can be or include a high-boiling pointliquid (solvent), such as described in U.S. Pat. Nos. 6,177,558 and6,337,393 to Brennan et al., each of which is incorporated herein byreference. In some embodiments, reagents can be configured to perform agas/vapor phase cleavage, as described, for example, in U.S. Pat. No.5,514,789 to Kempe, which is incorporated herein by reference.

Oligomers generally include any molecule formed of two or morecovalently linked subunits. The term oligomer, as used herein, also isintended to encompass polymers of any size or complexity. Accordingly,an oligomer can have any suitable number of subunits, for example,greater than ten, greater than one-hundred, or greater than one-thousandsubunits, among others. The various subunits of an oligomer can bestructurally identical (such as oligomers with a repeated subunit),structurally related but including distinct subunits (such as oligomersof different nucleotides or amino acids), and/or structurally unrelated(such as oligomers including different structural classes of subunits),as desired. Oligomers synthesized by the systems described herein canhave a predefined size (or length), composition, and/or sequence ofsubunits. However, such oligomers can be synthesized as mixtures ofoligomers, such as degenerate oligonucleotides synthesized with amixture of nucleotide components at one or more positions of theoligonucleotides.

The oligomers can be used for any suitable purpose(s). For example,nucleic acids oligomers can be used in genomics applications, such asgene expression analysis, detection of single-nucleotide polymorphisms,and/or high density TAQMAN assays, among others. Accordingly, nucleicacid oligomers can be used as probes (e.g., fluorescence in situhybridization (FISH) probes), primers (e.g., polymerase chain reaction(PCR) primers), substrates, test compounds for screens, and/or reagents,among others. Amino acid polymers similarly can be used as probes,primers, substrates (e.g., enzyme substrates such as kinase substrates),biological modulators, test compounds for screens, and/or reagents,among others.

III. Reagent Removal Mechanisms

Reagents, including reacted derivatives thereof, can be removed fromreaction compartments using one or more reagent removal mechanisms of anoligomer synthesis system. A reagent removal mechanism can removeexcess/unreacted reagent from a reaction compartment to a wastereservoir, such as an adjoining chamber, for example, as described abovein Section I. The reagent removal mechanism can be configured to removereagents from the reaction compartments of the array at substantiallythe same time or at different times for subsets of the array.Alternatively, or in addition, the reagent removal mechanism can beconfigured to move reagents within the array portion of a synthesissupport device, such as movement from a porous member to an adjoiningreaction compartment, among others.

The reagent removal mechanism can be configured to push and/or pullfluid from a reaction compartment. For example, the reagent removalmechanism can exert a positive pressure to push the fluid through thereaction compartment. Alternatively, or in addition, the reagent removalmechanism can exert a negative pressure to pull the fluid from thereaction compartment. The pressure exerted by the reagent removalmechanism can be adjustable and controlled by a processor to define therate of movement of reagents through a reaction compartment, time ofcontact with the reagents, etc. In some examples, reagents can beremoved by centrifugation of the synthesis support device.

The reagent removal mechanism can be configured to operate on onereaction compartment at a time, on a set of two or more reactioncompartments at the same time, or on all of the reaction compartmentsconcurrently. Accordingly, the reagent removal mechanism can be disposedadjacent each of the reaction compartments concurrently or can moveamong the reaction compartments of a synthesis support device, forexample by sliding back and forth and/or movement along two axes, amongothers, to selectively remove reagent(s) from a subset of the reactioncompartments.

IV. Examples

The following examples describe selected aspects and embodiments of thepresent teachings, including an exemplary synthesis system forsynthesizing oligomers in an array, support devices for oligomersynthesis in arrays, and methods of making and using such supportdevices. These examples and the various features and aspects thereof areincluded for illustration and are not intended to define or limit theentire scope of the present teachings.

Example 1 Exemplary System for Oligomer Synthesis in an Array

This example describes an exemplary system for oligomer synthesis in anarray; see FIG. 3.

Synthesis system 70 can include an array device 72, a reagent dispenser74, a flow controller 76, and a computing device 78, among others.Reagent dispenser 74 can dispense reagents to array device 72. Flowcontroller 76 can be disposed in fluid communication with the arraydevice for continuous or periodic removal of the dispensed reagents (orportions/derivatives thereof) from reaction compartments of the arraydevice. Computing device 78 can be disposed in communication with arraydevice 72, reagent dispenser 74, and/or flow controller 76 for operationand/or monitoring thereof.

Array device 72 can include a chamber structure or frame 80 and an arrayportion 82 held by the frame. Array portion 82 can define an array 84 ofselectively addressable reaction compartments 86. Frame 80 and arrayportion 82 can cooperatively define a chamber 88 in which fluid isreceived from the reaction compartments. Frame 80 can be connected totubing 90 that provides fluid communication between flow controller 76and chamber 88.

Reagent dispenser 74 can include a dispense head 92, reagents reservoirs94, and conduits 96 connecting the reagent reservoirs to the dispensehead. The dispense head 92 can include one or more dispense tips 98 fromwhich reagents 100 of the reagent reservoirs 94 can be dispensed, shownat 102. Dispense head 92 and/or tips 98 can be movable (such asorthogonally), shown at 104, to position the head or tips for selectivedelivery to reaction compartments 86. Alternatively, or in addition,array device 72 can be movable to position reaction compartments inrelation to dispense head 92 and/or tips 98.

Computing device 78 can control and coordinate operation of system 70.For example, computing device 78 can be configured to select a reagentreservoir(s) from which a reagent will be dispensed, to select aposition for head 92, and to operate a pump and/or valve(s) to controlthe volume of the reagent that is delivered. Computing device 78 canoperate flow controller 76 to control exposure of the reactioncompartments to reagents. Computing device 78 also or alternatively canbe in communication with one or more sensors of the system. The sensorscan be configured to sense any suitable aspects of the system, includingtemperature, reagent status, pressure, dispensed volume, reactionefficiency, etc.

Example 2 Exemplary Synthesis Support Device

This example describes an exemplary synthesis support device, andreaction compartments and reaction surfaces thereof; see FIGS. 4 and 5.Selected aspects of this synthesis support device were discussed abovein Example 1, particularly in the context of FIG. 3.

Support device 72 can include an array portion 82 formed by one or moreapposed layers of material. The apposed layers can include a porousmember 122, a channel layer 124, and a permeable retainer layer 126.

Porous member 122 can form a top layer of array portion 82. The porousmember can include a plurality of porous islands 128 separated by aspacer 130 to define array 84. The porous islands can have any suitablearrangement to define an array of addressable regions. The porousislands can be substantially more hydrophilic than the spacer, forexample, so that the spacer is hydrophobic and the islands hydrophilic.

Channel layer 124 can define an intermediate or lower layer of arrayportion 82. The channel layer can form an array of channels orthrough-holes 132 extending between opposing surfaces 134, 136 of thechannel layer. Channels 132 can correspond in number and arrangement tothe porous islands, so that channels 132 and porous islands 128 aresubstantially aligned and present in one-to-one correspondence. Eachchannel 132 can contain one more particles 138.

Retainer layer 126 can define a lower layer of array portion 82. Theretainer layer can be configured to retain particles 138 (and fluid) inchannels 132. The retainer layer also can be permeable to permit fluidflow through this layer. The retainer layer can be any permeablematerial, including a fiber filter (e.g., formed of glass fibers,cellulose, synthetic polymer strands, etc.), a layer of porous silicon,or the like.

Array portion 82 can be assembled from two or more layers, such as thesandwich of three layers shown herein, and can be supported by frame 80.Frame 80 can include a support flange 140 to support the array portionabove chamber 88. Frame 80 also can include an opposing flange 142 thatopposingly grips the array portion with support flange 140. Opposingflange 142 can be adjustable to restrict removal of array portion 82and/or separation or relative movement of the layers thereof duringoligomer synthesis. Opposing flange 142 can be removable to allowprocessing of the array portion during and/or after oligomer synthesis.

During oligomer synthesis, reagents can be addressed to each porousisland from dispense tips 98 of dispense head 92, shown at 144. Reagentscan be received by porous islands 128 and can flow to channels 132 forcontact with particles 138 therein. After reaction, the reagents (orportions/derivatives thereof) can be moved from channel 132, throughretainer layer 126 and into waste reservoir 88, shown at 146, to joinwaste fluid 148.

FIG. 5 shows an individual reaction compartment 170 of array portion 82of the synthesis support device 72. Reaction compartment 170 can beformed by porous island 128, channel 132, particles 138, or acombination thereof, among others.

Porous island 128 can include a plurality of pores 172, which are shownschematically in the present illustration. Individual pores or sets ofpores 172 can extend between opposing surfaces 174, 176 of porous member122 to permit passage of fluid through the island. Pores can be definedby pore walls 178 that include pore reaction surfaces 180. Pore reactionsurfaces 180 can include a first reactive moiety 182 covalently ornoncovalently coupled to the pore walls. Accordingly, pores 172 can beconfigured to create an island reaction sub-compartment 184.

Channel 132 can be configured to be defined by channel walls 186 havingchannel reaction surfaces 188. Channel reaction surfaces can include asecond reactive moiety 190 coupled to channel walls 186. In the presentillustration, second reactive moiety 190 is distinct from first reactivemoiety 182 of the porous member. In some examples, the same reactivemoiety can be coupled to each of the porous member and the channellayer, and/or a plurality of different reactive moieties can be includedin one or more of the surfaces. Channel 132 can form a channel reactionsub-compartment 192 adjoining the island reaction sub-compartment 184.

Channel reaction sub-compartment 192 can contain particles 138 havingparticle reaction surfaces 194. Particle reaction surfaces 194 caninclude a third reactive moiety 196, which can be the same or differentfrom the other reactive moieties. In the present illustration, thirdreactive moiety 196 is different from first and second reactive moieties182, 190. Use of different reactive moieties can provide support forsynthesis of different oligomers within a reaction compartment and/orselective uncoupling of a subset of an oligomer population from areaction compartment.

Example 3 Placement of Reagents in Reaction Compartments

This example describes exemplary methods of addressing reactioncompartments in fluid isolation or fluid communication; see FIG. 6. Inthe present sequence of configurations, first and second reagents 202,204 are shown being dispensed sequentially to reaction compartment 170with the first reagent in isolation from, and the second reagent incommunication with, other reaction compartments of array portion 82.

First reagent 202 can be dispensed from above (and/or adjacent) porousisland 128, shown at 206 in the first configuration. The first reagentcan be dispensed in a droplet(s) 208 having a volume insufficient tospread laterally beyond the island after contact with the porous island.Accordingly, the placed droplet, shown at 210 in the secondconfiguration, can be restricted substantially to the porous island andrestricted from flowing laterally over or into spacer 130 by adifference in surface energy of the porous island and the surroundingspacer. First reagent 202 thus can be received in reaction compartment170, in fluid isolation from other reaction compartments, shown at 212in the third configuration. The first reagent can be received by a forceor pressure exerted on the first reagent (such as a vacuum, a positivepressure, or a centrifugal force) and/or by capillary action.

Second reagent 204 can be dispensed to array portion 82 after the firstreagent, shown at 214 after placement, in the fourth configuration ofthe sequence. First reagent 202 can be substantially removed fromreaction compartment 170 before dispensing second reagent 204, as shownin the present illustration, or second reagent 204 can be dispensed toporous island 128 before substantial removal of first reagent 202 fromthe reaction compartment. The second reagent can be dispensed in a fluidvolume sufficient to spread laterally beyond the island, to “flood” theupper surface of the porous member, indicated at 216 in the fourthconfiguration. As a result, second reagent 204 can be introduced intoconcurrent contact with a plurality of the porous islands (and/or all ofthe porous islands) with a single dispensing operation. Furthermore, thesecond reagent can be received substantially concurrently in each of thereaction compartments of the array portion 82 from above the porousislands, shown for one of the reaction compartments at 218 in the fifthconfiguration. The second reagent can be removed after being received inthe reaction compartment, shown at 220 in the sixth configuration.

Example 4 Oligomer Release

This example describes exemplary methods of releasing oligomers fromvarious reaction surfaces of a support device; see FIG. 7.

Configuration 230 of the sequence shows completed oligomer populations232, 234, 236 connected, respectively, to support surfaces of porousmember 122, particles 138, and channel wall 186 of array portion 82.These oligomer populations can represent structurally identical ordistinct oligomer populations. A subset of the oligomer populations,such as oligomer populations 232 and 234, can be selectively uncoupled(cleaved) from their support surfaces by a cleavage treatment, indicatedat step 238. In the present illustration, step 238 includes a vaporphase cleavage/deprotection, without elution of the uncoupled oligomerpopulations. Accordingly, prior to cleavage, a subset of the oligomerpopulations, such as oligomer populations 232, 234, can be coupled totheir support surfaces using a different association mechanism than theremaining oligomer population(s), such as oligomer population 236.Configuration 240 shows oligomer populations 232, 234 uncoupled fromtheir support surfaces but not removed from regions adjacent thesesurfaces.

Porous member 122 can be separated from the channel and retainer layers124, 126, shown at step 242. The resultant configurations 244, 246 eachcan be disposed adjacent a sample holder 248, for example, a sampleholder having wells 250 or other receiving compartments arrangedaccording to the array of reaction sub-compartments of porous member 122and channel layer 124.

A force can be applied to each of the porous member and the channellayer, shown respectively at steps 252 and 254. The force can be appliedby a vacuum pump, pressurized gas, and/or by centrifugation, amongothers. The force can elute oligomer populations 232, 234 from theirrespective support structures into wells 250, shown in configurations256, 258. In some examples, additional fluid, such as an aqueous buffer,can be added to one or both reaction sub-compartments to facilitateelution of the oligomer populations from the porous member and channellayer. Eluted oligomers can be analyzed according to theirconcentration, purity, sequence, and/or the like, for example, forquality control purposes. Alternatively, or in addition, elutedoligomers can be used for any suitable assay(s). The eluted oligomersproduced by steps 252, 254 can form duplicate or corresponding arraysfor the same or different purposes.

Channel layer 124 can be separated from retainer layer 126 and particles138, shown at step 260 and represented by a partially disassembled statein configuration 262. Particles 138 can be discarded at this stage.

Configuration 264 shows oligomer population 236 being cleaved from itssupport surface by a laser 266 and subsequently analyzed. Oligomerpopulation 236 can be coupled to channel layer 124 by a linker 190 thatis resistant to the vapor phase cleavage treatment of step 238.Accordingly, oligomer population 236 can remain coupled to its supportsurface during the processing steps preceding step 260. Linker 190 canbe a photocleavable linker (e.g., o-nitro benzyl, among others), so thatlight 268 from laser 266 can be used to cleave the oligomer in a vacuum.The cleaved oligomer thus can be analyzed by travel through an electricfield by mass spectrometry, such as by matrix assisted laser desorptionionization (MALDI). Accordingly, channel layer 124 can be formed of aconductive material, such as silicon, among others.

Example 5 Fabrication of Support Devices

This example describes exemplary methods of fabricating support devicesfor synthesis of oligomers in an array; see FIGS. 8 and 9.

FIG. 8 is a series of views of a porous member being processed accordingto a method 270 of forming a porous member having an array ofhydrophilic islands and a hydrophobic spacer. Hydrophilic porous member272 can be formed from a nonporous member by any suitable treatment,such as chemical etching of a silicon wafer, among others, or may berendered porous by its fabrication (such as a fiber filter).

Step 274 can be performed by treatment of first porous member 272 with amodifying agent to create a hydrophobic porous member 276 from a morehydrophilic first porous member. In exemplary embodiments, step 274 canbe performed by treatment of a silicon porous member with a fluorosilaneor an alkane, among others. In some embodiments, step 274 can beperformed selectively, such as with a mask, to create hydrophilicislands and a hydrophobic spacer.

Step 278 can be performed next to add a first mask layer 280 tohydrophobic porous member 276. The first mask layer can be patterned ornot patterned, as shown in the present illustration. In some examples,step 278 can include forming a layer of a positive (or negative)photoresist on a surface of the hydrophobic porous member, such as byspin coating, among others.

Next, step 282 can place a second mask layer 284 on first mask layer 280to create assembly 286. Second mask layer 284 can be a pre-patternedmask layer, such as a quartz chromium mask layer having opticallytransparent and opaque regions 288, 290, respectively.

Step 292 can be performed next by exposure of assembly 286 to light 294.Light is permitted to pass through transparent regions 288 andrestricted from passage through opaque regions 290. Accordingly, firstmask layer 280 can be selectively exposed to the light according to thearrangement of transparent regions 288.

The second mask layer then can be removed and the first mask layerprocessed to selectively remove regions exposed (or not exposed) to thelight, shown at step 296, thereby creating uncovered regions 298.

Step 302 can be performed next to selectively modify uncovered regions298 of hydrophobic porous member 276 to create a patterned porous member304 having porous islands 306 and a hydrophobic spacer 308.

First mask layer 280 then can be removed, for example, by strippingphotoresist from patterned porous member 304, shown at step 310. Porousmember 304 then can be used for selective placement of reagents and/orto support oligomer synthesis in an array of reaction compartments.

Patterned porous member 304 can be fabricated by any suitable variationsof method 270. For example, a pre-patterned mask layer, such as a quartzchromium mask, can be placed directly onto hydrophobic porous member276, without use of a first mask layer. Next, hydrophobic porous member276 can be selectively ablated adjacent transparent positions of themask by exposure to light, such as ultraviolet light, to selectivelyincrease the hydrophilicity of regions of the porous member.

FIG. 9 is a series of views of structures produced by a method 320 offabricating a channel (permeable well) array. The channel array can beassembled with the patterned porous member produced by method 270 ofFIG. 8 to create an array portion of a synthesis support device. Thechannel array can include a substrate 322, such as a silicon substrate,that is patterned by method 320.

A first mask layer 324 can be applied to substrate 322, shown at step326. The first mask layer can be, for example, a positive or negativephotoresist.

A pre-patterned mask layer 328 then can be placed on first mask layer,shown at step 330, to form a substrate assembly 332. The pre-patternedmask layer can, for example, define a predefined spatial pattern ofpermissive and restrictive light transmission, such as with a quartzchromium mask.

Substrate assembly 332 can be exposed to light, shown at step 334. Thelight can selectively photolyze regions of the first mask layer apposedto transparent regions of pre-patterned mask layer 328 (see method 270of FIG. 8).

The pre-patterned mask layer 328 and photolyzed regions of the firstmask layer then can be removed, shown at step 336.

Substrate 322 then can be exposed to an etchant configured toselectively remove the substrate at unprotected regions 338 to createchannels 340 in a channel layer 342, shown at step 344. Any suitableetchant can be used including a chemical etchant (such as hydrofluoricacid), anodization (such as pulse anodization), and/or photoinduction,among others. Channels 340 can be through-holes of any suitable shape,include cylindrical, frustoconical, etc. Alternatively, channels 340 canbe recesses having a porous floor.

First mask layer 324 then can be removed and channels 340 modified tocreate derivatized channel layer 346, shown at step 348. Channelmodification can include reacting surfaces of the channels with a bisfunctional moiety (two or more reactive groups), for example, to coatthe channel surfaces with a reactive moiety. Next, the reactive moietyon the channel surface can be connected to a photocleavable linker bychemical reaction. Alternatively, a photocleavable linker can beselected that is directly reactive with the channel surfaces withoutprior modification using the bis functional moiety. A suitablephotocleavable linker can be stable during oligomer synthesis.

Channel layer 346 then can be further modified to create channelassembly 348, shown at step 350. In particular, a permeable retainerlayer 352 can be apposed to the channel layer to create permeable wells354. Optionally, particles 356 can be placed in the permeable wells andretained therein by retainer layer 352. Porous member 304 of method 270(see FIG. 8) can be placed over channel assembly 348 to form afiltration device defining an array of selectively addressable reactioncompartments.

Example 6 Further Aspects of the Present Teachings

This example suggests potential advantages of the synthesis platform ofthe present teachings over other platforms. These advantages can includeone or more of the following.

(1) Scalability: the synthesis scale can be defined by the dimensions ofthe well (or channel), the area of the solid support(s) (e.g., particlesurface area), and/or the concentration of reactive moieties on thesolid support(s).

(2) The number of oligomers produced per synthesis run can be defined bythe platform design (for example, the number of reaction compartmentsper synthesis support device).

(3) Cycle times can be directly related to the number of nozzles used todispense amidites (or other oligomer reagents) and the speed andaccuracy of dispensing.

(4) Reagent use can be minimized through surface tension localization.

(5) Substrates can be used as oligomer arrays where quality control canbe performed using fluorescence-based strategies.

(6) Arrays can be used for genomics applications.

(7) The platform can be compatible with various oligomer chemistries,such as peptide nucleic acids, locked nucleic acids, peptides, smallmolecules, and/or the like.

(8) Oligomers can be synthesized with a low cost per oligomer.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

1. A device for parallel synthesis of oligomers, comprising: a porousmember including an array of islands and a spacer joined to the array ofislands and separating the islands, the array of islands being morehydrophilic than the spacer, each island defining a plurality of porespermitting reagents to pass through such island; a channel structuredisposed adjacent the porous member and including a plurality ofreaction compartments disposed in one-to-one correspondence with thearray of islands; and a set of particles disposed in each reactioncompartment and retained therein by the channel structure, the set ofparticles being configured to support synthesis of at least one oligomerusing the reagents that pass through the corresponding island of thearray.
 2. A device for directing fluids, comprising: a porous memberincluding an array of islands and a spacer joined to the array ofislands and separating the islands, the islands and the spacer havingdifferent surface energies, the porous member including opposingsurfaces, each island defining a network of pores and being configuredto receive reagents and to permit passage of the reagents between theopposing surfaces.